Marijuana components, such as delta-9-tetrahydrocannabinol (THC), and endogenous cannabinoids such as anandamide (ANA) and 2-arachydonoylglycerol (2-AG) alter diverse immune functions. Two cannabinoid receptors have been discovered to date, the central cannabinoid receptor (CB1 receptor) and the peripheral cannabinoid receptor (CB2 receptor). The CB1 receptor is expressed predominantly in the central nervous system. The CB2 receptor is mainly expressed in in cells of the immune system, suggesting that the CB2 receptor is involved in immunoregulatory events. However, the role of cannabinoid ligands and their receptors in the immune system remains unknown. The objective of this research proposal is to investigete the role of CB2 receptor activation on specific T helper (Th) cells and macrophage functions. THC and other exogenous cannabinoids have been reported to inhibit the activity of Th1 cells (cells involved in call mediated immunity), and to enhance the activity of Th2 cells (cells involved in humoral activity). The effect of ANA and 2-AG on Th1/Th2 cell activation is still unknown. Interestingly, using splenocytes from wild type (CB2+/+) and CB2 receptor knockout mice (CB-/- mice), we have found that exogenous cannabinoids (nM concentrations) increase specific Th1 (intefleukin 2 (IL2) and interferon gamma/ (INFgamma) and Th2 (IL4) cytokine secretion via the CB2 receptor. Moreover, our findings also showed that the basal level of IL2 and IL4 is increased in CB2-/- splenocytes, suggesting that basal CB2 receptor activation modulates secretion of these two cytokines. In the present, we propose to look at the in vivo effect of cannabinoid receptor activation on Th1/Th2 cell activation using CB2+/+ and CB-/- mice. The mice will be treated with cannabinoids and their splenocytes analyzed for Th1 (IL2 and INFgamma) and Th2 cytokines (IL4, IL5, and IL10). Since Th2 cell activation induces humoral response, antibody production will be measured in antigen challenged splenocytes derived from the cannabinoid treated mice. To assess Th deficiency, we want to analyze T cell populations from CB2+/+ and CB2-/- mice by fluorescence activated cell sorting. Cannabinoids are also known to affect macrophage cytokine production. We have found that CB2 receptor activation modulates lipopolysaccharide (LPS)-induced tumor necrosis alpha (TNFalpha) production. TNFalpha is decreased in macrophages derived from CB2-/- mice. We now want to include IL1beta, IL6, and IL10 cytokine analysis. Since TNFalpha is known to be involved in apoptosis, we also want to investigate whether CB2 receptor activation plays a role in TNFalpha-mediated apoptosis by measuring caspase-1 and -8 in CB2+/+ and CB2-/- macrophages. Finally, it is known that LPS can induce TNFalpha production via signaling mechanisms that include the mitogen activated protein kinase (MAPK) pathway. It is also known that cannabinoids can activate the MAPK pathway in macrophages. Therefore, we want to determine whether CB2 receptor modulation of this pathway is responsible for the decrease in TNFalpha we observed. We will use MAPK inhibitors in CB2+/+ and CB2-/- macrophages and measure TNFalpha production. Completion of the present proposal will help us learn about the role of CB2 receptor activation on the immune system. It will also help us elucidate the effects of ANA and 2-AG in these events. Findings from this project will be clinically useful, since it will help elucidate the use of cannabinoids to alter specific immune functions without having any unwanted CNS side effects.